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Method for synthesizing lixisenatide

A technology of lixisenatide and side chain protecting groups, which is applied in the field of solid-phase synthesis of polypeptides, can solve the problems of difficult acquisition of specific strains, consumption of manpower and material resources, and inapplicability to large-scale industrial production.

Inactive Publication Date: 2012-07-11
HYBIO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, gene recombination is mostly used in foreign countries to prepare lixisenatide, but specific strains are not easy to obtain, the consumption of manpower and material resources is large, the purification is difficult, and the product purity is low, which is not suitable for industrialized large-scale production.

Method used

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  • Method for synthesizing lixisenatide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Preparation of Fmoc-Lys(Boc)-Rink Amide Resin:

[0054] Weigh 10g of Fmoc-Rink Amide Resin with a substitution degree of 0.25mmol / g, put it into a solid-phase reaction column, wash the resin twice with DMF (100ml+50ml), soak the resin in 50ml of DMF for 45 minutes to fully swell the resin, add 20% 40ml of piperidine / DMF solution, reacted for 10 minutes, drained the reaction liquid, and washed the resin 6 times with DMF (50ml×6). Weigh 3.52g of Fmoc-Lys(Boc)-OH, 1.01g of HOBt, and 1.39ml of DIC, dissolve them in 40ml of DMF, stir slowly in an ice bath for 3 minutes, pour into a solid-phase reaction column and mix with the resin, and react at room temperature for 2 hours. The Ninhydrin method was used to detect the completion of the reaction, the reaction solution was removed, and the resin was washed with DMF three times (50m×6) to obtain Fmoc-Lys(Boc)-Rink Amide Resin. The molar ratio of Fmoc-Lys(Boc)-OH:HOBt:DIC used in the above operations is 1:1:1.2, wherein the mol...

Embodiment 2

[0071] Preparation of Fmoc-Lys(Boc)-Rink Amide-BHA Resin:

[0072]Weigh 20g of Fmoc-Rink Amide-BHA Resin with a substitution degree of 0.15mmol / g, put it into a solid-phase reaction column, wash the resin twice with DMF (180ml+100ml), then soak it in DMF for 60 minutes to fully swell the resin, add 80ml of 20% piperidine / DMF solution was reacted for 10 minutes, the reaction solution was drained, and the resin was washed 6 times with DMF (100ml×6). Weigh 7.03g of Fmoc-Lys(Boc)-OH, 2.78ml of DIC, dissolve in 80ml of DMF, pour the Fmoc-Lys(Boc)-OH and DIC solution into the solid phase reaction column and mix with the resin, react at room temperature for 2 hours, Ninhydrin method After detecting the completion of the reaction, the reaction solution was removed, and the resin was washed three times with DMF (100m×6) to obtain Fmoc-Lys(Boc)-Rink Amide-BHAResin. The molar ratio of Fmoc-Lys(Boc)-OH:DIC used in the above operations is 1:1.2, wherein the molar number of Fmoc-Lys(Boc)-O...

Embodiment 3

[0089] Preparation of Fmoc-Lys(Boc)-Rink Amide-MBHA Resin:

[0090] Weigh 10g of Fmoc-Rink Amide-MBHA Resin with a substitution degree of 0.25mmol / g, put it into a solid-phase reaction column, wash the resin twice with DMF (100ml+50ml), soak the resin in 50ml of DMF for 45 minutes to fully swell the resin, add 20% piperidine / DMF solution 40ml, reacted for 10 minutes, drained the reaction solution, and washed the resin 6 times with DMF (50ml×6). Weigh 3.52g of Fmoc-Lys(Boc)-OH, 1.01g of HOBt, and 1.39ml of DIC, dissolve in 40ml of DMF, stir slowly in ice bath for 3 minutes, pour into solid phase reaction column and mix with resin, and react at room temperature for 2 hours. The Ninhydrin method was used to detect the completion of the reaction, the reaction solution was removed, and the resin was washed with DMF three times (50m×6) to obtain Fmoc-Lys(Boc)-RinkAmide-MBHA Resin. The molar ratio of Fmoc-Lys(Boc)-OH used in the above operation:HOBt:DIC is 1:1:1.2, wherein the molar...

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Abstract

The invention relates to a polypeptide synthesis field in solid phase, and particularly relates to a method for synthesizing lixisenatide. The method uses amino resin as a solid phase carrier, comprising: subsequently gradually amino acids by solid phase peptide connecting method of Fmoc protection strategy after coupling Fmoc-Lys(Boc)-OH on the resin, to obtain complete protection peptide resin, and then cutting the peptide resin by a lysate, settling the peptide resin in ether, centrifugating the peptide resin and drying the peptide resin to obtain the crude product of lixisenatide, purifying the crude product by a reversed phase HPLC, and then salinizing the purified crude product to obtain lixisenatide. The method has advantages of simple operation and good yield, and satisfies the industrialization requirement.

Description

technical field [0001] The invention relates to the field of solid phase synthesis of polypeptides, in particular to a method for synthesizing lixisenatide. Background technique [0002] Lixisenatide is a linear polypeptide containing 44 amino acid residues, its molecular formula is: C 215 h 347 N 61 o 65 S, molecular weight: 4858.55, CAS number: 320367-13-3. The structure of lixisenatide is based on the modification of the carbon terminal of the exendin-4 molecule, and six lysine residues are connected sequentially. The amino acid sequence is shown in SEQ ID NO:1. [0003] Lixisenatide is a peptide drug for the treatment of type 2 diabetes. It is currently in the phase III clinical research stage. It is a glucagon-like peptide 1 (GLP) developed by Antoine Sanofi and Zealand Pharmaceuticals. -1) Receptor agonist, lixisenatide (lixisenatide) combined with basal insulin in the treatment of patients with type 2 diabetes can significantly improve blood sugar control, reduce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/575C07K1/20C07K1/06C07K1/04
CPCY02P20/55
Inventor 刘飞付信刘建马亚平袁建成
Owner HYBIO PHARMA
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